Abstract
The effects of reaction conditions on the spectrophotometric and fluorometric assays using alternative substrates (p-nitrophenyl palmitate and 4-methylumbelliferyl oleate) were investigated to optimize them for the high-throughput screening of lipase activity from agricultural products. Four model lipases from Chromobacterium viscosum, Pseudomonas fluorescens, Sus scrofa pancreas, and wheat germ (Triticum aestivum) were allowed to hydrolyze the alternative substrates at different substrate concentrations (1–5 mM), operating pH (5.0–8.0), and operating temperatures (25–55°C). The results show that both the spectrophotometric and fluorometric assays worked well at the standard reaction conditions (pH 7.0 and 30°C) for finding a typical lipase, although pH conditions should be considered to detect the catalytic activity of lipases, which are applicable to more acidic or alkaline pH circumstances. To validate the optimized conditions, the high-throughput screening of lipase activity was conducted using 17 domestic agricultural products. A pileus of Pleurotus eryngii showed the highest activity in both the spectrophotometric (633.42 μU/mg) and fluorometric (101.77 μU/mg) assays. The results of this research provide practical information for the high-throughput screening of lipases using alternative substrates on microplates.
Highlights
Lipases are carboxylic ester hydrolases found in diverse organisms, including animals, plants, fungi, and bacteria that can catalyze the hydrolysis of triacylglycerols into diacylglycerols, monoacylglycerol, and free fatty acids [1]. ese enzymes are highly soluble in aqueous phases, similar to other enzymes; their substrates are highly water-insoluble acylglycerol species, and lipases can efficiently catalyze hydrolysis at the interface between water and oil droplets [2]
At a specific enzyme concentration, the reaction rate is relatively slow in the range of low substrate concentration, whereas it gradually increases with the substrate concentration until substrate saturation point is reached [24]. us, highthroughput screening for catalytic activity should consider whether the target substrate added to the reaction medium is enough to react with the target enzyme
There was a reduction of lipase activity at higher substrate concentrations, a phenomenon called substrate inhibition that is generally observed in lipasecatalyzed hydrolysis
Summary
Lipases (i.e., triacylglycerol hydrolase, EC 3.1.1.3) are carboxylic ester hydrolases found in diverse organisms, including animals, plants, fungi, and bacteria that can catalyze the hydrolysis of triacylglycerols into diacylglycerols, monoacylglycerol, and free fatty acids [1]. ese enzymes are highly soluble in aqueous phases, similar to other enzymes; their substrates are highly water-insoluble acylglycerol species, and lipases can efficiently catalyze hydrolysis at the interface between water and oil droplets [2]. Lipases typically exhibit unique selectivities on their substrates, such as typoselectivity, regioselectivity, and stereoselectivity [5, 6], though a few lipases show promiscuous behavior toward their substrates [7] Because of those attractive properties, lipases are being studied in the fields of selective hydrolysis for flavor/texture improvement [8, 9], esterification for the synthesis of structured lipids or functional compounds [10, 11], and other catalytic reactions. Using original acylglycerol species as substrates for lipase reactions is a hindrance to high-throughput screening because most of the relevant methodologies are complicated or laborious due to their high lipophilicity and analytical difficulty of their products
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